This work aimed to evaluate the artificial lighting strategies to increase the viability of microalgae biodiesel production. Different long-term, frequency, and short photoperiods were examined. Besides, the productivities of the process, the chemical composition of the biomass, biodiesel quality, and energy balance were assessed. The results showed that Scenedesmus obliquus CPCC05 can store sufficient energy to sustain cell growth for continuous periods of up to 2 h in the dark, without affecting the productivities of the process. The values for the maximum biomass (63.88 mg/L h) and lipid (18.9 mg/L h) productivities as well for the calorific value (20.4 kJ/g) were obtained at a photoperiod of 24 t/d. It was qualitatively observed that the photoperiod significantly influenced the fatty acid profile of single-cell oil and, consequently, the quality of the produced biodiesel. Finally, the use of photoperiods also proved to be an effective strategy to improve the energy balance and reduce the environmental impact of the microalgae biodiesel production process.